The overall objective is to establish the mechanisms responsible for the regulation of the oxidation of branched-chain amino acids by mammalian cells. Studies will be conducted with purified branched-chain Alpha-ketoacid dehydrogenase (BCKDH) complex, purified BCKDH kinase, purified BCKDH phosphatase, isolated hepatocytes, isolated myocytes, perfused rat heart, isolated liver and heart mitochondria, and intact animals. The working hypothesis is that covalent modification by phosphorylation-dephosphorylation is an important mechanism for regulation of BCKDH. Studies will be conducted to further characterize the BCKDH complex purified in this laboratory from rabbit liver, to isolate and characterize the phosphatase responsible for dephosphorylation and activation of BCKDH to further investigate with intact animals the effects of various nutritional and hormonal states as well as specific drugs on the activity and phosphorylation state of BCKDH in various tissues, and finally to determine with isolated hepatocytes, myocytes, and mitochondria whether the phosphorylation state of the BCKDH is subject to acute hormonal control. The detailed specific aims include to establish (a) molecular weight and subunit organization of BCKDH; (b) the number and amino acid sequences of the phosphorylation sites in the 47,000 Mr Alpha-subunit; (c) the effectiveness of various Alpha-chloro- and Alpha-ketoacids as inhibitors of BCKDH kinase; (d) the role of Ca++ as an inhibitor of BCKDH kinase; (e) the effects of fasting, obesity, diabetes, and protein deprivation on the phosphorylation state of BCKDH in liver, heart, muscle, kidney, and adipose tissue; and (f) whether the phosphorylation state of BCKDH is subject to hormonal control. These studies should help explain altered branched-chaim amino acid metabolism known to occur in numerous diseases and metabolic conditions, including diabetes, obesity, maple syrup urine disease, phenylketonuria, kwashiorkor, and cancer.